Sofosbuvir metabolite, 2′-deoxy-2′-fluoro-2′-C-methyluridine (PSI-6206) was studied for the first time by surface enhanced Raman spectroscopy (SERS) using the paper-based SERS substrate. The quantification limit of PSI-6206 by SERS was found to be 13 ng L−1 (R2 value = 0.959, RSD = 5.23%). For the structural and quantitative analysis of PSI-6206 in blood plasma, an interference-free HPLC-SERS method was developed and compared to HPLC-DAD and HPLC–MS methods. The SERS quantification of the drug by the paper substrate was 4 orders of magnitude more sensitive than that by the diode array detector. In addition, the SERS detection provided unique structural identification of the drug in blood plasma, similar to Mass spectroscopy detector. Due to the disposable nature of the SERS substrate, the new method does not suffer from the known “memory effect” which is known to lead to false positive identification in traditional HPLC-SERS methods. Therefore, the presented HPLC-paper SERS platform holds great potential for the sensitive and cost effective determination of drugs and their metabolites in biological fluids.